The present invention relates to the cytochrome P-450 (CYP) family of heme proteins, which mediate metabolic processes. More specifically, the present invention relates to polynucleotides encoding a novel CYP family member, the CYP3AX protein, and variants thereof. Further, the present invention relates to methods for identifying and obtaining drug candidates and inhibitors for therapy of disorders related to the malfunction of CYP3AX encoding genes, as well as to methods of diagnosing the status of such disorders.
Members of the cytochrome P-450 (CYP) family of hemoproteins metabolize a wide variety of endogenous substrates and xenobiotics including carcinogens, toxins and drugs (Daly, Toxicol. Lett. 102-103 (1998), 143-7; Touw, Drug Metabol. Drug Interact. 14 (1997), 55-82). Of the human CYP proteins, members of the CYP3A subfamily are of major importance, since collectively they form the largest portion of all human CYP isoforms. The human CYP3A subfamily consists of three homologous proteins encoded by distinct genes (CYP3A4, CYP3A5 and CYP3A7) (Thummel, Annu. Rev. Pharmacol. Toxicol. 38 (1998), 389-430). The pharmacological significance of CYP3A is due to its expression in all major organs contributing to drug disposition (gastrointestinal tract, liver, kidney) and to its remarkably broad substrate spectrum. Based on the available experimental data it is estimated that between 45% and 60% of currently used drugs are substrates for CYP3A (Li, Toxicology 104 (1995), 1-8; Evans, Science 286 (1999), 487-91). The substrates of CYP3A include substances as diverse as steroids, antidepressants, benzodiazepines, immunosuppressive agents, imidazole antimycotics, macrolide antibiotics and toxins. The high homology among the CYP3A proteins and the available experimental data have led to the assumption that the three CYP3A isoforms have similar substrate spectra; however, some studies indicate the possibility of differences (Thummel, Annu. Rev. Pharmacol. Toxicol. 38 (1998), 389-430).
A considerable variation in the content and catalytic activity of CYP3A has been described in the general population. For example, the activities of the CYP3A4 protein in liver biopsies often vary up to 40-fold (Westlind, Biochem. Biophys. Res. Commun. 259 (1999), 201-5; Shimada, J. Pharmacol. Exp. Ther. 270 (1994), 414-23). Human in vivo studies have also indicated considerable interindividual variability in CYP3A4 activity, but its extent has been smaller. The reason for this discrepancy is not clear, but it could reflect the poor CYP3A isozyme specificity of the substrates used (Thummel, Annu. Rev. Pharmacol. Toxicol. 38 (1998), 389-430). CYP3A5 exhibits a similar variability of expression. In adult Caucasians, the CYP3A5 mRNA and protein were detected in the liver of 10 to 30% of samples, while the protein was found in the kidney and intestine of 70% subjects (Jounaidi, Biochem. Biophys. Res. Commun. 221 (1996), 466-70) and references therein). CYP3A7, the third CYP3A isoform, was originally isolated from fetal liver, and was subsequently found in 54% of liver samples in adults (Schuetz, Pharmacogenetics 4 (1994), 11-20).
The variability of CYP3A expression, coupled with the broad spectrum of drugs that are metabolized by CYP3A proteins, creates a potential for potentially harmful drug interactions involving these isozymes in patients undergoing therapies with multiple drugs (Thummel, Annu. Rev. Pharmacol. Toxicol. 38 (1998), 389-430). In addition, the interindividual variation in the CYP3A activity could also influence the individual predisposition to cancers caused by environmental carcinogens. For example, CYP3A proteins metabolize aflatoxin B1 (Wang, Biochemistry 37 (1998), 12536-45), a mycotoxin strongly implicated in the etiology of liver cancer, which is a major cause of premature death in many areas of Africa and Asia (Henry, Science 286 (1999), 2453-4). Forrester et al. (Proc. Natl. Acad. Sci. U S A 87 (1990), 8306-10) found that the rates of metabolic activation of aflatoxin B1 correlated with the level of CYP3A proteins in microsomes. It has also been proposed that high levels of CYP3A in humans could predispose an individual to cancer risk from bioactivated tobacco-smoke procarcinogens (Paolini, Nature 398 (1999), 760-1).
Clearly, there is a need for a better understanding of the factors underlying the variability of CYP3A expression and its effects on drug metabolism and drug efficacy. Such improved understanding of CYP3A family member structure, function and expression should lead to an optimization of therapies with drugs, for example in cancer treatment. The present invention fulfills this need and provides other related advantages.
It is an aspect of the present invention to provide a polynucleotide encoding a cytochrome P450 (CYP) 3AX polypeptide or a biologically active fragment thereof, that is (a) a polynucleotide encoding a polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8 10 or 12; (b) a polynucleotide encoding a polypeptide, the polynucleotide comprising a coding sequence as depicted in any one of SEQ ID NOS: 1, 3, 5, 7, 9 or 11; (c)a polynucleotide encoding a polypeptide derived from the polypeptide encoded by a polynucleotide of (a) or (b) by way of substitution, deletion or addition of one or more amino acids of the amino acid sequence encoded by the polynucleotide of (a) or (b); (d) a polynucleotide the complementary strand of which hybridizes under moderately stringent conditions with a polynucleotide of any one of (a) to (c); (e) a polynucleotide encoding a polypeptide the sequence of which has an identity of at least 80% to the amino acid sequence of the polypeptide encoded by a polynucleotide of any one of (a) to (d); (f) a polynucleotide encoding a fragment or an epitope-bearing portion of a polypeptide encoded by a polynucleotide of any one of (a) to (e); (g) a polynucleotide encoding an epitope-bearing portion of a CYP3AX polypeptide comprising amino acid residues from about 405 to about 425 in SEQ ID NO: 2; (h) a polynucleotide comprising at least 15 nucleotides of a polynucleotide of any one of (a) to (g); (i) a polynucleotide of any one of (a) to (d), wherein at least one nucleotide is deleted, added or substituted and wherein the nucleotide deletion, substitution and/or addition results in an altered expression or activity of the CYP3AX polypeptide; (j) a polynucleotide encoding a molecular variant of the polypeptide encoded by the polynucleotide of (a) or (b); (k) a polynucleotide of (j), wherein the methionine at position 275 in SEQ ID NO. 2 is mutated; (1) a polynucleotide encoding a polypeptide that is immunospecifically recognized by an antibody that has been elicited by immunization with a polypeptide encoded by a polynucleotide of (a) or (b); (m) a polynucleotide which hybridizes under stringent conditions with a probe having the sequence of the polynucleotide of (a) or (b), or a fragment thereof; or (n) a polynucleotide the nucleotide sequence of which is a variant of the nucleotide sequence of a polynucleotide of any one of (a) to (m), due to genetic code degeneracy, or a complementary sequence thereto; provided that the polynucleotide does not consist of the nucleotide sequence set forth in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38.
In certain embodiments the polynucleotide is DNA, in certain embodiments it is genomic DNA and in certain embodiments it is RNA. In certain embodiments the polynucleotide is operatively linked to an expression control sequence. In certain other embodiments the invention relates to a vector comprising any one of the just described polynucleotides, and in certain other embodiments the invention relates to a host cell comprising any one of the just described polynucleotides or the just described vector.
It is another aspect of the invention to provide a method for producing a CYP3AX polypeptide or fragment thereof comprising (a) culturing the above described host cell under conditions and a time sufficient to permit expression of the polypeptide; and (b) isolating the protein or fragment from the culture. In other embodiments the invention provides a method for producing cells capable of expressing the CYP3AX polypeptide comprising genetically engineering cells with the any one of the just described polynucleotides or the just described vector.
In other embodiments the invention provides a CYP3AX protein or fragment thereof that is a polypeptide encoded by any one of the above described polynucleotides, a polypeptide produced by the method just described, or a polypeptide expressed by cells produced according to the method just described. In another embodiment the invention provides a gene encoding the CYP3AX protein, an in another embodiment there is provided an antibody which binds specifically to the CYP3AX protein. In other embodiments the invention provides a nucleic acid molecule or a vector comprising a nucleic acid sequence that is complementary to any one of the above described polynucleotides, and in other embodiments there is provided a nucleic acid molecule capable of specifically recognizing and cleaving any one of the above described polynucleotides, or a vector comprising such a nucleic acid molecule.
Turning to another aspect of the invention, there is provided a transgenic non-human animal comprising at least one of the above described polynucleotides, vectors or genes. In certain further embodiments, the animal comprises at least one inactivated wild type allele of the CYP3AX gene. In certain embodiments, the animal is a mouse or a rat.
In still another aspect, the invention provides a method of identifying and obtaining a CYP3AX inhibitor or activator capable of modulating the activity of the CYP3AX gene or the gene product thereof comprising the steps of (a) contacting the CYP3AX protein described above or a cell expressing the CYP3AX gene described above or comprising any one of the above described polynucleotides in the presence of components capable of providing a detectable signal in response to drug metabolism, with a compound to be screened under conditions that permit CYP3AX-mediated drug metabolism, and (b) detecting the presence or absence of a signal or increase of a signal generated from the metabolized drug, wherein the presence or increase of the signal is indicative for a putative inhibitor or activator. Thus, in particular embodiments the invention provides a method of identifying and obtaining a CYP3AX inhibitor or activator capable of modulating the activity of a CYP3AX gene or a gene product thereof, comprising the steps of: (a) contacting, in the presence of at least one component capable of providing a detectable signal in response to drug metabolism, either (i) the CYP3AX protein described above, or (ii) a cell expressing a CYP3AX gene or comprising a CYP3AX polynucleotide described above, in the absence and presence of a candidate compound under conditions and for a time sufficient to permit CYP3AX-mediated drug metabolism; and (b) comparing a level of the detectable signal provided in the absence of the agent to a level of the detectable signal provided in the presence of the agent, wherein an altered (i.e., increased or decreased in a statistically significant manner) signal level is indicative of an inhibitor or activator of a CYP3AX gene or of a CYP3AX gene product. In certain further embodiments the cell is a host cell as described above, or a CYP3AX producing cell obtained by the above described method, or is present in the transgenic non-human animal described above.
In another embodiment there is provided a method of identifying and obtaining an CYP3AX inhibitor or activator capable of modulating the activity of the CYP3AX gene or the gene product thereof comprising the steps of (a) contacting the CYP3AX protein of claim 10 with a first molecule known to be bound by the protein to form a first complex of the protein and the first molecule; (b) contacting the first complex with a candidate compound to be screened; and (c) measuring whether the compound displaces the first molecule from the first complex. In one embodiment, the measuring step comprises measuring the formation of a second complex of the protein and the compound, and in certain further embodiments the measuring step comprises measuring the amount of the first molecule that is not bound to the protein. In certain further embodiments the first molecule is cyclosporin, midazolam, lovastatin, nifedipin, diltiazem, erythromycin, lidocaine, amiodarone, or taxol. In other further embodiments, the first molecule is detectably labeled.
The present invention also provides a method for identifying a molecular variant of CYP3AX comprising (a) determining the presence or the level of any one of the above described polynucleotides in a sample from a subject; (b) determining the presence or the level of a CYP3AX protein; and (c) determining the presence of a mutation in the polynucleotide. In one embodiment the invention provides a method for identifying a molecular variant of CYP3AX comprising determining, in a sample from a subject, a level selected from the group consisting of (a) a level of the CYP3AX polynucleotide described above; (b) a level of a CYP3AX protein or fragment thereof as also described above; and (c) a level of the presence of a mutation in at least one of the above described CYP3AX polynucleotides. In a further embodiment the invention provides a method for diagnosing or prognosing a disorder related to the expression of a molecular variant CYP3AX gene, or susceptibility to such a disorder comprising the step of determining the level of drug metabolism. In certain embodiments the disorder is cancer. In certain other embodiments the method comprises PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques, hybridization techniques or immunoassays.
In certain other embodiments there is provided a method for identifying a molecular variant of CYP3AX comprising determining, in a sample from a subject, (a) a level of the CYP3AX polynucleotide described above; (b) a level of a CYP3AX protein or fragment thereof as described above; or (c) a level of the presence of a mutation in the CYP3AX polynucleotide described above. In a related embodiment there is provided a method for determining, in a subject, a risk for having or being susceptible to, the presence of, or the prognosis of a disorder related to the expression of a molecular variant CYP3AX gene, comprising the steps of determining, in a sample from the subject, (1) a level of drug metabolism; and (2) a level selected from the group consisting of (a) a level of the CYP3AX polynucleotide described above; (b) a level of a CYP3AX protein or fragment thereof described above; and (c) a level of the presence of a mutation in the CYP3AX polynucleotide described above. In certain further embodiments, said disorder is cancer. In certain other further embodiments, at least one level is determined by PCR, ligase chain reaction, restriction nuclease digestion, direct nucleotide sequencing, a nucleic acid amplification technique, a nucleic acid hybridization technique or an immunoassay. In another embodiment there is provided a method for treating a disorder related to the expression of a molecular variant CYP3AX gene in a subject, comprising (a) identifying the disorder according to any one of the methods just described; and (b) administering to the subject a medicament to abolish or alleviate said disorder, where the selection and dosage of such medicament will be known to a person having ordinary skill in the art based upon the disclosure herein.
In certain other embodiments the method further comprises administering to a subject a medicament to abolish or alleviate the disorder. In certain other further embodiments there is provided a method for the production of a pharmaceutical composition comprising synthesizing the compound identified and obtained in the method or a derivative thereof in a pharmaceutically acceptable form. In another further embodiment the method comprises formulating a drug or pro-drug in the form suitable for therapeutic application and preventing or ameliorating the disorder of the subject diagnosed in the just described methods. In a further embodiment the compound drug or prodrug is a derivative of a medicament administered as described above. In another embodiment the invention relates to an inhibitor or activator identified or obtainable by any of the above described methods. In a further embodiment the inhibitor or activator binds specifically to the above described CYP3AX protein.
Turning to another embodiment, the invention provides a method for the detection of any one of the above described CYP3AX polynucleotides, or a method for genotyping of individual CYP3AX alleles or variants. In certain embodiments, the polynucleotide is a CYP3AX polynucleotide as described above or a complementary nucleic acid molecule thereto or a ribozyme. In one embodiment the oligonucleotide is about 15 to 50 nucleotides in length, which in certain further embodiments is an oligonucleotide. In another embodiment the invention provides a method of detecting a CYP3AX protein comprising detection of a CYP3AX binding antibody as described above, detection of a CYP3AX binding protein, detection of the expression of a CYP3AX gene comprising a CYP3AX polynucleotide or detection of a CYP3AX allelic variant. In certain further embodiments, the method of detecting a CYP3AX protein further comprises distinguishing two or more CYP3AX alleles or variants.
In other embodiments the invention is directed to the use of an effective dose of a drug or prodrug for the preparation of a pharmaceutical composition for the treatment or prevention of a disorder of a subject comprising a CYP3AX polynucleotide, wherein in certain further embodiments the disorder is cancer.